1. Field of the Invention
The present invention relates to a new C-terminal .alpha.-amidating enzyme preparation and a process for production and use thereof.
2. Description of the Related Art
A number of biologically active peptides isolated from neural or endocrine tissues have an .alpha.-amide structure at their carboxyl termini (C-termini). In most cases, the presence of the C-terminal .alpha.-amide structure is essential for their biological activity. Therefore, the C-terminal .alpha.-amide formation of the peptide is an important factor for in vivo activation of prohormones into active mature hormones. The recent elucidation of the nucleotides sequences of many precursors of .alpha.-amidated peptides shown that the amino acid residue that is .alpha.-amidated in the nature peptide is necessarily followed by a glycyl residue in the precursor. In procine pituitary, Bradburg, A. F. et al, Nature 298, 686-688, 1982, first characterized the .alpha.-amidating activity converting a synthetic substrate D-Tyr-Val-Gly to D-Tyr-Val-NH.sub.2 and demonstrated that the C-terminal glycine in the substrate serves as a nitrogen-donor for .alpha.-amidation.
Because of the importance to clarify the mechanism of .alpha.-amide formation in tissues and of the promising usefulness of the enzyme for the production of C-terminally .alpha.-amidated peptides using, for example, recombinant DNA techniques, many attempts to purify the enzyme have been done but the engyme has not so far been obtained in a pure state. Eipper et al, Proc. Natl. Acad. Sci. US, 80, 5144-5148, 1983, reported that the .alpha.-amidating enzyme derived from pituitary gland requires copper cation and ascorbate for its activity. Husain, I. et al, FEBS Lett., 152 227-281, 1983; and Kizer, J. S. et al, Proc. Natl. Acad. Sci. US, 81, 3228-3232, 1984, also reported a C-terminal .alpha.-amidating enzyme, but did not report a purified enzyme. Recently, Murthy A. S. N. et al, J. Biol. Chem., 261, 1815-1822, partially purified a C-terminal amidating enzyme from pituitary gland of cattle, and showed that some types of enzymes having different molecular weights and electric charges are present. However, no type of enzyme has been homogeneously purified.